We have developed orthotopic xenograft transplants from human colon cancer cell lines that recapitulate the metastatic pattern of colon cancer in patients and have made the novel observation that rescue of attenuated TGF[unreadable] receptor expression inhibits metastatic capability in these models even though invasion at the primary tumor site was maintained. Moreover, we showed genetic attenuation of TGF[unreadable] receptor expression in a model that was poorly metastatic generated robust metastasis to the liver and lungs. These results are significant because they raise the possibility that regeneration of attenuated TGF[unreadable] signaling would be an effective target in the treatment of metastases. Development of effective treatments for metastatic disease would have a large impact since metastases are the major cause of death in solid cancers. A key to capitalizing on the repression of metastases TGF[unreadable] signaling is the elucidation of the mechanism by which metastasis is inhibited. The mechanistic basis of this project is our novel observation that TGF[unreadable] mediates control of cell death through a mechanism involving the activation of PKA (independent of cAMP, but AKAP dependent) that leads to proteosomal degradation of survivin and XIAP. These IAPs have been shown to be critical to aberrant cell survival signaling in tumor models (Dohi et al, 2007). PKA initiates a multi-functional cascade directed at disruption of this cell survival mechanism. Aims I and II will focus on the characterization of this pathway. Specific Aim I will address the hypothesis that Smad 2 plays a critical role in recruitment of the survivin/XIAP complex to the vicinity of the activated PKA. There are two PKA regulatory subunit isoforms designated PKAI and PKAII. We have found that poorly metastatic cells have a preponderance of RII while the isogenic complementary highly metastatic cells have high RI. This suggests that the regulatory subunits function differently from each other with respect to the efficiency of TGF[unreadable]/PKA pathway activation and that TGF[unreadable] signaling affects the expression of the isoforms as well. Thus these 2 hypotheses will also be tested in Aim I. Our previous work noted that in addition to repression of survivin, AKT was inhibited by TGF[unreadable] signaling. Since AKT dephosphorylation is carried out by PP2A (another AKAP directed enzyme) and is a PKA substrate, the hypothesis that PP2A activation is part of the TGF[unreadable]/PKA pathway and plays a dual role countering cell survival through promotion of proteasomal stimulation and ensuing degradation of XIAP will be tested in Aim II. vivo. Due to the critical role of the survivin/XIAP complex in controlling cell survival in vitro, we will test the hypothesis that mutations which stabilize the complex will enhance metastatic colonization in poorly metastatic cells and to block regression of established metastases by TGF[unreadable] receptor reconstitution in highly metastatic cells in Specific Aim III. The specific aims of the project are: I. Determine the mechanism of TGF[unreadable] activation of PKA;II. Determine the potential role of PP2A in the TGF[unreadable]/PKA pathway. III. Test the hypothesis that the survivin/XIAP complex is essential to metastatic spread. PUBLIC HEALTH RELEVANCE: One of the most pressing problems in cancer treatment is the lack of effective therapies for advanced metastatic disease. The elucidation of metastatic mechanisms and the validation that their disruption results in the regression of the metastases will be addressed in this project. We have identified a candidate mechanism for inappropriate cell survival signaling in metastases that will be subjected to validation and evaluation as a therapeutic target for the treatment of metastases in this project.